The vascular endothelium is recognized as the principle source of tissue plasminogen activator (tPA) in blood and therefore must play a major role in determining the fibrinolytic state of the vascular system. Cultured human endothelial cells (HEC) continue to produce a PA which is immunochemically similar to the tPA isolated from human plasma. Characterization of the HEC-tPA has shown that it has a molecular weight of 100,000, it is inactive in its native state, and it does not bind to fibrin clots. Preliminary studies on the molecular state of the HEC-tPA have suggested that it is a complex consisting of a 60,000 dalton anti-tPA immunoprecipitable component and an undefined 40,000 dalton inhibitor. Because endothelial cell cultures offer a useful model to define the contribution of the vascular endothelium in fibrinolysis it is proposed to initially characterize the tPA-containing complex released by primary cultures of HEC and then begin to define the mechanisms by which HEC-derived plasma PAs are regulated. Using cultured human umbilical vein endothelial cells I propose to purify the 100,000 dalton complex, determine the nature of the interaction between the tPA and its putative inhibitor, the site and timing of complex formation, and the identity and specificity of the inhibitor. Once the complex has been characterized studies will focus on the regulation of tPA production by plasma components. These studies will define the precise mechanisms by which the HEC-derived tPA and its modulated by inhibitors and the mechanisms by which cellular PA production is regulated. It is anticipated the proposed studies will have clinical application in determining potential therapeutic procedures for modulating the firbrinolytic state.